This invention relates to immunotoxin conjugates and their use to selectively delete a target population of cells. More particularly, the present invention relates to the utilization of a toxin B chain moiety coupled to a cell surface affinity binding agent to potentiate the cytotoxicity provided by a cell surface affinity binding agent coupled to a toxin A chain moiety.
Ricin is one of a number of plant proteins which, in minute quantities, exhibits considerable toxicity toward eukaryotic cells. Ricin is composed of two glycoprotein chains covalently linked via a single disulfide bond. The A chain of ricin, having an apparent molecular weight (AMW) of about 30,000, is responsible for the expression of toxicity, and acts enzymatically upon the 60S ribosomal subunit leading to irreversible abrogation of protein synthesis [Olsnes, et al., FEBS Lett 28, 48-50 (1972)]. Ricin B chain (AMW 32,000) functions as a lectin with specificity for galactose and serves to bind the toxin to the plasma membrane [see, for example, Baenziger, et al., J. Biol. Chem. 254, 9795-9799 (1979)]
The use of ricin, or the purified ricin A chain, in conjunction with antibodies, has been the subject of great interest as potentially useful reagents in tumor therapy. Antibody-ricin and antibody-A chain conjugates, or "immunotoxins", have been used in a number of systems with varying degrees of success [see, for example, Vitetta, et al., Science 219, 644-650 (1983); Thorpe, et al., Immunol. Rev 62, 120-158 (1982); Neville, et al., Immunol. Rev. 62, 75-91 (1982); and Jansen, et al., Immunol. Rev. 62, 185-216 (1982)].
Procedures for deleting selected populations of cells by ricin A chain-antibody conjugates are well recognized. The antibodies of choice are those which react with antigens on tumor cells or on subsets of normal lymphocytes. By deletion of the tumor cells, it is possible, for example, to reduce tumor burdens in vivo [Krolick, et al., J. Exp. Med. 155, 1797 (1982)] and to remove tumor cells from bone marrow for autologous marrow transplantation [Thorpe, et al., Nature (London) 271, 752 (1978); and Krolick, et al., Nature (London) 295 604 (1982)].
By deletion of normal subsets of lymphocytes, it is possible to "up" or "down" regulate the immune response. The advantage of immunotoxins is that they are highly selective in their target cell specificity and that small doses can eliminate unwanted cells. Ricin A chain-antibody conjugates have been used primarily to delete normal and neoplastic B cells, both in vivo and in vitro. Certain laboratories have also used conjugates of ricin A chain and monoclonal antibody to eliminate neoplastic cells of T cell origin and a variety of other cancerous cells.
However, ricin A chain-antibody conjugates are not active when used against certain types of tumor cells (e.g., some T cell tumors) [Neville, et al., Immunol. Rev. 62, 75 (1982); and Thorpe, et al., Immunol. Rev. 62, 119 (1982)].
In contrast, immunotoxins coupled to the whole ricin toxin are much more potent cytocidal agents. Unfortunately, the presence of the galactose binding site of ricin B in intact ricin prevents its use in vivo since its target cell specificity thereby is lost. Attempts to overcome the nonspecificity of ricin-containing immunotoxins by blocking the galactose binding site are ongoing; however, their use in vivo has to date not been described.
Others have described studies in which ricin A chain-antibody conjugates can be potentiated by the addition of free B chain to cell cultures (Neville et al., supra). It has been postulated, therefore, that the B chain of ricin plays two functional roles: (1) to facilitate entry of ricin into the cell by virtue of its galactose-binding properties, and (2) to allow the A chain to gain rapid access to the cytoplasm, perhaps by formation of a pore in the endocyticvesicle membrane.
In a recent unpublished study, applicants have discovered that injection of mice with nontoxic ricin A chain followed 4-8 hours later by injection with non-toxic ricin B chain produces ricin-induced death. This probably occurs by reformation of the intact ricin molecule in the serum or on the surface of circulating cells. It is therefore believed that the B chain plays an active role in potentiating the toxic activity of the ricin A chain.